African trypanosomiasis, also called African sleeping sickness, infects tens of thousands of individuals yearly in endemic areas, and is accompanied by continuing high social and economic cost. Existing treatments for trypanosomiasis are woefully inadequate, due both to the emergence of drug resistance that results in high treatment failures rates (30% in some areas), and high toxicity resulting in significant morbidity (10%) and drug-related mortality (5%). There thus remains an outstanding need for rational design of new trypanocidal therapies, particularly those that minimize host toxicity by targeting unique trypanosome biology. Glucose metabolism is the sole source of ATP for the infectious lifecycle stage of the African trypanosome, Trypanosoma brucei, and enzymes central to sugar metabolism are housed in the glycosome, an organelle not found in the parasite's mammalian host(s). Hence, both glycosome function and the control mechanisms governing enzyme activity inside the glycosome are important targets for drug design. We have demonstrated that ATP production in this organism is sensitive to environmentally-influenced changes in glycosomal solution conditions, including pH. Characterizing the intraglycosomal environment is therefore a necessary step in understanding essential glycolytic pathways, and would lay the groundwork for development of anti-trypanosome therapies that target control of glucose metabolism. However, this information is currently lacking at the most basic level. Neither pH nor glucose has been quantified inside the glycosome and their environmentally influenced dynamic range(s) are unknown, a paucity that reflects the historical lack of methodologies to allow quantitative intraglycosomal measurement. Here we propose development of peptide-targeted small molecule and recombinant protein-based sensors to quantitatively determine intraglycosomal pH and glucose levels. Our preliminary data indicates that such sensors can be delivered to the glycosomes of live parasites. Developed sensors can be subsequently modified for measurement of other glycosomal solutes, including ATP, and used to investigate control of glycolysis as a response to other potentially important environmental and developmental conditions, including glucose and divalent salt concentrations, nutrient depletion, and calcium signaling. Resulting findings will illuminate the mechanisms of dynamic regulation of the glycosomal environment, reveal conditions that influence the activity of this essential metabolic pathway, and introduce methodologies likely facilitate a series of new approaches to understanding and testing parasite metabolism. Notably, techniques pioneered in this study can be extended to analysis of other pathogenic kinetoplastid parasites, such as Trypanasoma cruzi and Leishmania spp. that also localize ATP production in glycosomes. In addition, the methodologies can be modified to evaluate the intraorganellar environment in other important subcellular compartments such as the mitochondria, endoplasmic reticulum, and Golgi apparatus. The work is therefore likely to have impact(s) beyond African trypanosomiasis.